Title: Cholinergic transmission in C. elegans: Functions, diversity, and maturation of ACh-activated ion channels Treinin M, Jin Y Ref: Journal of Neurochemistry, 158:1274, 2021 : PubMed
Acetylcholine is an abundant neurotransmitter in all animals. Effects of acetylcholine are excitatory, inhibitory, or modulatory depending on the receptor and cell type. Research using the nematode C. elegans has made ground-breaking contributions to the mechanistic understanding of cholinergic transmission. Powerful genetic screens for behavioral mutants or for responses to pharmacological reagents identified the core cellular machinery for synaptic transmission. Pharmacological reagents that perturb acetylcholine-mediated processes led to the discovery and also uncovered the composition and regulators of acetylcholine-activated channels and receptors. From a combination of electrophysiological and molecular cellular studies, we have gained a profound understanding of cholinergic signaling at the levels of synapses, neural circuits, and animal behaviors. This review will begin with a historical overview, then cover in-depth current knowledge on acetylcholine-activated ionotropic receptors, mechanisms regulating their functional expression and their functions in regulating locomotion.
BACKGROUND: To survive dynamic environments, it is essential for all animals to appropriately modulate their behavior in response to various stimulus intensities. For instance, the nematode Caenorhabditis elegans suppresses the rate of egg-laying in response to intense mechanical stimuli, in a manner dependent on the mechanosensory neurons FLP and PVD. We have found that the unilaterally placed single interneuron ALA acted as a high-threshold mechanosensor, and that it was required for this protective behavioral response. RESULTS: ALA was required for the inhibition of egg-laying in response to a strong (picking-like) mechanical stimulus, characteristic of routine handling of the animals. Moreover, ALA did not respond physiologically to less intense touch stimuli, but exhibited distinct physiological responses to anterior and posterior picking-like touch, suggesting that it could distinguish between spatially separated stimuli. These responses required neither neurotransmitter nor neuropeptide release from potential upstream neurons. In contrast, the long, bilaterally symmetric processes of ALA itself were required for producing its physiological responses; when they were severed, responses to stimuli administered between the cut and the cell body were unaffected, while responses to stimuli administered posterior to the cut were abolished. CONCLUSION: C. elegans neurons are typically classified into three major groups: sensory neurons with specialized sensory dendrites, interneurons, and motoneurons with neuromuscular junctions. Our findings suggest that ALA can autonomously sense intense touch and is thus a dual-function neuron, i.e., an interneuron as well as a novel high-threshold mechanosensor.
Title: Locomotion analysis identifies roles of mechanosensory neurons in governing locomotion dynamics of C. elegans Cohen E, Yemini E, Schafer W, Feitelson DG, Treinin M Ref: J Exp Biol, 215:3639, 2012 : PubMed
The simple and well-characterized nervous system of C. elegans facilitates the analysis of mechanisms controlling behavior. Locomotion is a major behavioral output governed by multiple external and internal signals. Here, we examined the roles of low- and high-threshold mechanosensors in locomotion, using high-resolution and detailed analysis of locomotion and its dynamics. This analysis revealed a new role for touch receptor neurons in suppressing an intrinsic direction bias of locomotion. We also examined the response to noxious mechanical stimuli, which was found to involve several locomotion properties and to last several minutes. Effects on different locomotion properties have different half-lives and depend on different, partly overlapping sets of sensory neurons. PVD and FLP, high-threshold mechanosensors, play a major role in some of these responses. Overall, our results demonstrate the power of detailed, prolonged and high-resolution analysis of locomotion and locomotion dynamics in enabling better understanding of gene and neuron function.
BACKGROUND: Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single-neuron mRNA profiling of PVD. RESULTS: Selectively photoactivating PVD, FLP, and downstream interneurons via Channelrhodopsin-2 (ChR2) enabled the functional dissection of this nociceptive network, without interfering signals by other mechanoreceptors. Forward or reverse escape behaviors were determined by PVD and FLP, via integration by command interneurons. To identify mediators of PVD function, acting downstream of primary nocisensor molecules, we knocked down PVD-specific transcripts by RNAi and quantified light-evoked PVD-dependent behavior. Cell-specific disruption of synaptobrevin or voltage-gated Ca(2+) channels (VGCCs) showed that PVD signals chemically to command interneurons. Knocking down the DEG/ENaC channel ASIC-1 and the TRPM channel GTL-1 indicated that ASIC-1 may extend PVD's dynamic range and that GTL-1 may amplify its signals. These channels act cell autonomously in PVD, downstream of primary mechanosensory molecules. CONCLUSIONS: Our work implicates TRPM channels in modifying excitability of and DEG/ENaCs in potentiating signal output from a mechano-nociceptor neuron. ASIC-1 and GTL-1 homologs, if functionally conserved, may denote valid targets for novel analgesics.
The mechanisms controlling the formation and maintenance of neuronal trees are poorly understood. We examined the dynamic development of two arborized mechanoreceptor neurons (PVDs) required for reception of strong mechanical stimuli in Caenorhabditis elegans. The PVDs elaborated dendritic trees comprising structural units we call "menorahs." We studied how the number, structure, and function of menorahs were maintained. EFF-1, an essential protein mediating cell fusion, acted autonomously in the PVDs to trim developing menorahs. eff-1 mutants displayed hyperbranched, disorganized menorahs. Overexpression of EFF-1 in the PVD reduced branching. Neuronal pruning appeared to involve EFF-1-dependent branch retraction and neurite-neurite autofusion. Thus, EFF-1 activities may act as a quality control mechanism during the sculpting of dendritic trees.
Title: The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors Biala Y, Liewald JF, Ben-Ami HC, Gottschalk A, Treinin M Ref: Mol Biology of the cell, 20:1419, 2009 : PubMed
RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation.
RIC-3 belongs to a conserved family of proteins influencing maturation of nicotinic acetylcholine receptors (nAChRs). RIC-3 homologues were shown to differently affect different nAChRs. Here we show that coexpression with RIC-3 increases the level of surface expression of DEG-3 while slightly reducing the level of surface expression of DES-2, both subunits of the DEG-3/DES-2 nAChRs. Those different effects are a likely explanation for the previously demonstrated effects of RIC-3, an endoplasmic reticulum resident protein, on properties of this receptor. To understand how RIC-3 interacts with different nAChR subunits, we identified and characterized domains and residues enabling this interaction. This analysis shows that conserved residues in the second RIC-3 transmembrane domain are needed for its interactions with two different Caenorhabditis elegans nAChRs, DEG-3/DES-2 and ACR-16. These conserved residues do not, however, function alone; rather, we show that additional domains also enable RIC-3's interactions with these receptors. Interestingly, the relative importance of these residues or of other domains mediating interactions of RIC-3 with nAChRs differs for the two different receptors. Differences in the way that RIC-3, predicted to be an intrinsically disordered protein, interacts with different receptors and receptor subunits suggest that it may adopt different conformations to enable these interactions. Such differences may explain both the effects of RIC-3 on receptor properties and the differences in its effects on different receptors.
Title: The BTB-MATH protein BATH-42 interacts with RIC-3 to regulate maturation of nicotinic acetylcholine receptors Shteingauz A, Cohen E, Biala Y, Treinin M Ref: Journal of Cell Science, 122:807, 2009 : PubMed
RIC-3 is a member of a conserved family of proteins that affect nicotinic acetylcholine receptor maturation. In yeast and in vitro, BATH-42, a BTB- and MATH-domain-containing protein, interacts with RIC-3. BATH-42 is also known to interact with the CUL-3 ubiquitin ligase complex. Loss of BATH-42 function leads to increased RIC-3 expression and decreased activity of nicotinic acetylcholine receptors in Caenorhabditis elegans vulva muscles. Increased expression of RIC-3 is deleterious for activity and distribution of nicotinic acetylcholine receptors, and thus the effects of BATH-42 loss of function on RIC-3 expression explain the associated reduction in receptor activity. Overexpression of BATH-42 is also detrimental to nicotinic acetylcholine receptor function, leading to decreased pharyngeal pumping. This effect depends on the C-terminus of RIC-3 and on CUL-3. Thus, our work suggests that BATH-42 targets RIC-3 to degradation via CUL-3-mediated ubiquitylation. This demonstrates the importance of regulation of RIC-3 levels, and identifies a mechanism that protects cells from the deleterious effects of excess RIC-3.
Title: RIC-3 and nicotinic acetylcholine receptors: biogenesis, properties, and diversity Treinin M Ref: Biotechnol J, 3:1539, 2008 : PubMed
Nicotinic acetylcholine receptors (nAChRs) belong to a diverse and widely expressed family of ion channels. These receptors are pentamers assembled from multiple combinations of subunits, with different subunit compositions producing receptors having different properties and functions. The diverse functions of nAChRs include an essential role in excitation of skeletal muscles and many modulatory roles throughout the central nervous system. Nicotinic receptors are also implicated in a number of brain pathologies such as epilepsy, schizophrenia, and Alzheimer's disease. Thus, it is important to understand the cellular mechanisms controlling both the numbers and the properties of surface expressed nAChRs. Genetic analysis in Caenorhabditis elegans identified a number of proteins specifically needed for biogenesis of nAChRs. Among these proteins is RIC-3, a member of a family of proteins having conserved structure and function. RIC-3 influences both surface expression and properties of nAChRs and its effects are subtype specific. Here we suggest that receptor-specific chaperones such as RIC-3 may play important roles in controlling receptor diversity by selectively regulating surface expression of nAChRs having specific subunit compositions.
Title: RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains Ben-Ami HC, Yassin L, Farah H, Michaeli A, Eshel M, Treinin M Ref: Journal of Biological Chemistry, 280:28053, 2005 : PubMed
Members of the RIC-3 gene family are effectors of nicotinic acetylcholine receptor (nAChR) expression in vertebrates and invertebrates. In Caenorhabditis elegans RIC-3 is needed for functional expression of multiple nAChRs, including the DEG-3/DES-2 nAChR. Effects of RIC-3 on DEG-3/DES-2 functional expression are found in vivo and following heterologous expression in Xenopus leavis oocytes. We now show that in X. leavis oocytes RIC-3 also affects the kinetics and agonist affinity properties of the DEG-3/DES-2 receptor. Because these effects are mimicked by increasing the ratio of DEG-3 subunits within DEG-3/DES-2 receptors, this suggests that RIC-3 may preferentially promote maturation of DEG-3-rich receptors. Indeed, effects of RIC-3 on functional expression of DEG-3/DES-2 positively correlate with the DEG-3 to DES-2 ratio. All RIC-3 family members have two transmembrane domains followed by one or two coiled-coil domains. Here we show that the effects of RIC-3 on functional expression and on receptor properties are mediated by the transmembrane domains and do not require the coiled-coil domains. In agreement with this, mammals express a RIC-3 transcript lacking the coiled-coil domain that is capable of promoting DEG-3/DES-2 functional expression. Last, we show that RIC-3 affects DEG-3 quantity, suggesting stabilization of receptors or receptor intermediates by RIC-3. Together our results suggest that subunit-specific interactions of RIC-3 with nAChR subunits, mediated by the transmembrane domains, are sufficient for the effects of RIC-3 on nAChR quantity and quality.
Title: Genetic dissection of an acetylcholine receptor involved in neuronal degeneration. Treinin M, Halevi S, Yassin L Ref: Cholinergic Mechanisms, CRC Press, :207, 2004 : PubMed
Title: Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression Halevi S, Yassin L, Eshel M, Sala F, Sala S, Criado M, Treinin M Ref: Journal of Biological Chemistry, 278:34411, 2003 : PubMed
In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat alpha 7, and human alpha 7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human alpha 4 beta 2 and alpha 3 beta 4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT3 serotonin receptors, whereas it barely modified alpha1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo.
Mutations in ric-3 (resistant to inhibitors of cholinesterase) suppress the neuronal degenerations caused by a gain of function mutation in the Caenorhabditis elegans DEG-3 acetylcholine receptor. RIC-3 is a novel protein with two transmembrane domains and extensive coiled-coil domains. It is expressed in both muscles and neurons, and the protein is concentrated within the cell bodies. We demonstrate that RIC-3 is required for the function of at least four nicotinic acetylcholine receptors. However, GABA and glutamate receptors expressed in the same cells are unaffected. In ric-3 mutants, the DEG-3 receptor accumulates in the cell body instead of in the cell processes. Moreover, co-expression of ric-3 in Xenopus laevis oocytes enhances the activity of the C.elegans DEG-3/DES-2 and of the rat alpha-7 acetylcholine receptors. Together, these data suggest that RIC-3 is specifically required for the maturation of acetylcholine receptors.
Title: Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation Yassin L, Samson AO, Halevi S, Eshel M, Treinin M Ref: Biochemistry, 41:12329, 2002 : PubMed
The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking.
Title: Characterization of the deg-3/des-2 receptor: a nicotinic acetylcholine receptor that mutates to cause neuronal degeneration Yassin L, Gillo B, Kahan T, Halevi S, Eshel M, Treinin M Ref: Molecular & Cellular Neurosciences, 17:589, 2001 : PubMed
The nicotinic acetylcholine receptor family (nAChR) is a large family of acetylcholine-gated cation channels. Here we characterize the Caenorhabditis elegans DEG-3/DES-2 nAChR, a receptor identified due to its involvement in neuronal degeneration. Pharmacological analysis of a DEG-3/DES-2 receptor expressed in Xenopus oocytes shows that this receptor is preferentially activated by choline. This choline sensitivity of the DEG-3/DES-2 channel can explain its role in neuronal degeneration, as shown by the toxic effects of choline on oocytes expressing the mutant DEG-3/DES-2 channel. We also show that in C. elegans the DEG-3/DES-2 receptor is localized to nonsynaptic regions, including the sensory endings of chemosensory neurons. This localization is in agreement with a role for this receptor in chemosensation of choline, as inferred from a defect in chemotaxis for choline seen in deg-3 mutants. Thus, this work also provides evidence for the diversity of nonsynaptic activities associated with nAChRs.
Title: Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon Treinin M, Gillo B, Liebman L, Chalfie M Ref: Proc Natl Acad Sci U S A, 95:15492, 1998 : PubMed
The deg-3 gene from the nematode Caenorhabditis elegans encodes an alpha subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an alpha subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.
Title: A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans Treinin M, Chalfie M Ref: Neuron, 14:871, 1995 : PubMed
Neurotoxicity through abnormal activation of membrane channels is a potential cause of neurodegenerative disease. Here we show that a gain-of-function mutation, deg.3(u662), leads to the degeneration of a small set of neurons in the nematode C. elegans. The deg.3 gene encodes a nicotinic acetylcholine receptor alpha subunit, which in the region of transmembrane domain II is most similar to the neuronal alpha 7 subunits from rat and chicken. The u662 mutation changes a residue in the second transmembrane domain, the domain thought to form the channel pore. A similar change in the equivalent amino acid in the chick protein produces channels that desensitize slowly. Channel hyperactivity may underlie the degenerations seen in the C. elegans deg.3(u662) mutants, since antagonists of nicotinic acetylcholine receptors suppress the deg-3(u662) mutant phenotypes.
